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Expression of structure-specific recognition protein mRNA in fetal kidney and Fe-nitrilotriacetate-induced renal carcinoma in the rat
CancerLetters 106(1996) 271-278
Expression of structure-specific recognition protein mRNA in fetal kidney and Fe-nitrilotriacetate-induced renal carcinoma in the rat Yun-Yan Xianga, Dong-Yu Wanga, Masamitsu Tanakaa, Hisaki Igarashia, Yasuhisa Naitoa. Yoshihisa Ohtawarab, Qiong Shenc, Haruhiko Sugimuraa,* “First Department of Pathology, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 43 l-31, Japan bDepartment of Urology, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-31, Japan ‘Departmeni qf Precancerous Studies, Henan Medical Universiry Zhengzhou, Henan 4.50052, People $ Republic, of China
Received22April 1996;revision received24 May 1996;accepted24 May 1996
Abstract
Specificexpressionof the structure-specificrecognitionprotein(SSRP)genewasinvestigatedin rat fetal, adult, and tumor tissuesusinga 2.0-kb partial sequence of rat SSRPcDNA isolatedfrom a cDNA library of rat renalcell carcinoma.The resultsrevealedthat it was rather specifically expressedin rat fetal kidney and renal cell carcinomainduced by Fenitrilotriacetate,but not in adult kidney,whenvariousorgansweretestedby Northernblot analysis.In situ hybridizationfurther demonstrated that it waslocatedin the neoplasticcellsof renalcell carcinomaand in the epithelialcellsof fetal kidney but undetectablein any cellsof normaladultkidney. Theseobservations seemto imply the involvementof SSRPgene,which is believedto recognizestructuralalterationsof DNA, in kidney developmentand carcinogenesis of certaintypes of kidney cancer. Keywords:
Rat kidney; Fetaldevelopment;Renalcell neoplasm;Structure-specificrecognitionprotein
1. Introdection Nitrilotriacetate (NTA) is an effective metalchelating agent used as a substitute for polyphosphates in laundry detergents [ 1,2]. Because chronic exposure to high levels of NTA producestoxicity and neopiasmsin the urinary tract [3], an experimental model of renal adenocarcinoma was established by repeated intraperitoneal injections of ferric nitrilotriacetate (Fe-NTA), an iron chelate of NTA that in* Corresponding author.Tel: +81 53 4352220;fax: +81 53 4352225.
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duces renal proximal tubular necrosis which in turn leads to a high incidence (@I-92%) of renal adenocarcinoma in male rats and mice [4,5]. The molecular changes in DNA induced by FeNTA have been investigated, and Fe-NTA has been found to induce DNA single- and double-strand breaks in the presence of reductant in vitro [6]. In recent years, the renal toxicity of Fe-NTA has come to be attributed to mediation by reactive oxygen species (ROS) [7-101 such as hydroxyl ra.dicals (OH) and hydrogen peroxide (H202). However, the only information available on altered gene expression in rat renal adenocarcinoma induced by Fe-NTA has
0 1996 Elsevier Science Ireland Ltd. All rights reserved
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been the overexpression of K-cadherin reported by us previously [l 11. In the present study, we obtained a cDNA clone that encodes the rat structure-specific recognition protein (SSRP) by screening a cDNA library of Fe-NTA-induced rat renal cell carcinoma. The expressions of rat SSRP in various rat organs were investigated by Northern blot analysis and in situ hybridization to see whether they are involved in kidney development and carcinogenesis. 2. Materials
and methods
2. I. Cell lines and tissues The transplantable tumor cell line RRCC-1 was derived from an AC1 rat renal cell carcinoma induced by Fe-NTA according to the method of Okada et al. [ 121. Other transplantable tumor cell lines, S4, derived from spontaneously occurring gastric adenocarcinoma of Wistar-Furth rats, and m731 and m673 derived from choriocarcinoma of Wistar-Furth rats were obtained from Dr. M. Miyamoto, Department of Pathology, Osaka University Medical School, Japan. Cells were maintained as described [ 111. Tissue from esophageal tumors induced by precursors of Nnitrososarcosine ethyl ester was obtained from Wistar rats [ 131. Tissue from various organs was obtained from day 20 fetal Wistar rats and 5-week-old Wistar rats. All of the samples were immediately frozen in liquid nitrogen and stored at -80°C until use.
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(T.Tn) obtained from the Japanese Cancer Research Resources Bank (JCRB). The hD-8 clone, cloned at the EcoRI site of the plasmid pBluescript SK(+) (Stratagene, LA Jolla, CA), is about 1.3 kb in size and extends from bp 725 to 2063 of the published human SSRPl nucleotide sequence [ 161 (Fig. 1). Northern blot hybridization analysis showed high level expression of hD-8 in the rat renal cell carcinoma RRCC-1. In the present study, therefore, the hD-8 cDNA clone was used as a probe for screening the RRCC-1 cDNA library to isolate rat cDNA for SSRP. To make the probe, plasmid DNA was digested with EcoRI and purified on a 1% agarose gel. 2.4. cDNA library construction
and screening
The rat RRCC-1 cDNA library was synthesized from 5 ,ug of cell line RRCC- 1 poly(A)+ RNA by the cDNA synthesis system plus an oligodeoxythymidylate primer (Amersham, Buckinghamshire, UK). The size-fractionated cDNA was ligated to ;Igt 10 arms and then packaged into phage particles with Gigapack II Gold Packaging Extract (Stratagene, LA Jolla, CA). The filters, prepared from the RRCC-I cDNA library, were screened with [a-32P]dCTPlabeled hD-8 cDNA probe. After hybridization, the filters were washed under a high stringent condition [ 141 and autoradiographed for 3 days. The cDNA clones isolated from the library were subcloned into the pGEM-T plasmid vector (Promega, Madison, WI).
2.2. RNA preparation 2.5. DNA sequencing Total RNAs of the cell lines, tumor tissues and various rat organs were extracted by guanidiniuml cesium chloride ultracentrifugation. The subsequent poly(A)+ RNA was then selected from total RNA by oligodeoxythymidylate cellulose chromatography [141. 2.3. Probe for screening In the previous study, we isolated a human D-8 cDNA clone (hD-8) from a human cDNA library by differential screening according to the method described previously [15]. The human cDNA library was established from a squamous cell carcinoma cell line (T.T) and its transplanted tumor in a nude mouse
Nucleotide sequences of the cDNA clones obtained from the RRCC-1 library were determined by the double-stranded dideoxy-chain termination method using T7 DNA polymerase (Sequenase; Version 2.0, United States Biochemical Corp., Cleveland, OH). A computer-assisted homology search was carried out by the FASTA program in the GenBank and EMBL databases using an on-line system to DDBJ (1111 Yata, Mishima, Japan). 2.6. Northern blot analysis Rat total RNAs (1Opg each) from various cell lines and tissues were separated in denaturing aga-
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rose gels and transferred to nitrocellulose membranes. Hybridization was carried out, followed by high stringent washing and autoradiography. Hybridization and washing conditions were as described by Sambrook et al. [ 141. 2.7. In situ hybridization In situ hybridization on cryostat sections was conducted as described previously [ll]. The rSSRP1 sense and antisense RNA probes were produced by in vitro transcription from linearized pGEM-T plasmids containing the entire rSSRP1 cDNA in both orientations in the presence of digoxigenin-labeled UTP (Boehringer Mannheim, Mannheim, Germany) using T3 or I7 RNA polymerase, according to the manufacturer’s instructions. Final washing conditions and detection of the hybridization signal were as described in [ 111. 3. Results 3.1. Isolation of a cDNA clone for SSRP A total of 3.5 X IO5 recombinant phages were screened with the initial human D-8 clone as a probe to isolate rat cDNA for SSRP. Three positive clones, which were demonstrated to be identical to each other by restriction enzyme mapping, were obtained after three rounds of purification. Computer-assisted comparison of the DNA sequences revealed one of them to be 100% homologous to the published SSRP cDNA sequences of rat CIIDBP-r [ 171 (Fig. 1, rCIhSSRP1
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Fig. 1. Diagrammatic nucleotide sequence comparison of rSSRP1 and hD-8 with known rat CIIDBP-r [17] and human SSRPl [16], respectively, and corresponding nucleotide positions. ICIIDBP, rat CIIDBP-r; hSSRP1, human SSRPl. The open boxes represent open reading fragments and the hatched box refers to the position of the HMG box in these nucleotide sequences.
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27.3
IDBP), which extends from 1 to 1353 bp of the whole CIIDBP-r sequence and contains 645 bp of additional 5’ sequencethat had 70% homology with the human SSRPl [16] and 94% homology with murine T160 [18]. Becauseit is isolated by using the partial sequenceof human SSRPl as a probe, we refer to the clone in this study as rat SSRPl (rSSRP1) (Fig. 1). 3.2. Expression of rSSRP1 mRNA in various rat tissues The mRNA Eevel of rSSRP1 was determined in various organs of fetal and adult rats using the entire length of rSSRP1 cDNA as a probe (Fig. 2). Total RNAs were isolated from the brain, heart, lung, stomach, intestine, colon, kidney, spleen, liver, skin, testis, ovary and uterus of 5-week-old Wistar rats and/or day 20 rat embryos. In the day 20 embryos, rSSRP1 was found to be strongly expressed in the brain, either heart or lung, stomach, spleen and kidney with a major transcript size of 2.9 kb (Fig. 2A, Lanes l-3, 6, 7). A low level of expression was also present in the liver, skin and placenta (Fig. 2A, Lanes4, 5, 8, 9). In adults, rSSRP1 was strongly expressedin the testis (Fig. 2B, Lane 11) and weakly expressed in the brain, heart, lung, forestomach, glandular stomach, colon, spleen, ovary and uterus (Fig. 2B, Lanes l-5, 7, 9, 12, 13)” but there was no expression in the intestine, kidney or liver {Fig. 2B, Lanes 6, 8, 10). Among the various organs investigated, tissuespecificity was only found in the kidney. rSSRP1 was strongly expressed in fetal kidney, but not expressed in adult kidney. Levels of rSSRP1 mRNA in neoplasmswere investigated by using kidney, esophagus,stomach and uterine tumor tissues. rSSRP1 was expressed in rat kidney transplantable tumor line RRCC-1 at the sametranscript size as in fetal kidney but not in adult kidney (Fig. 2C, Lanes 4, 8-10) whereasrSSRP1 expression was observed in both the cancer and normal tissuesof other organs including the esophagus(Fig. 2C. Lanes 5-7) stomach (Fig. 2B, Lanes4, 5, and C, Lane l), and uterus (Fig. 2B, Lane 13, and C, Lanes2, 3) 3.3.
In
situ hybridization of rSSRP1 in rat kidney
The results of in situ hybridization in rat day 17
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Fig. 2. Northern blot analysis of rSSRP1 in rat tissues. Total RNA (1Opg of each) was extracted from various organs of day 20 fetal Wistar rats (A), 5-week-old Wistar rats (B), and rat cancer tissues and cell lines (C). (A) Lane I, brain; Lane 2, heart and lung; Lane 3, stomach; Lane 4, intestine; Lane 5, liver; Lane 6, spleen; Lane 7, kidney; Lane 8, skin; and Lane 9, placenta. (B) Lane I, brain; Lane 2, heart; Lane 3, lung; Lane 4, forestomach; Lane S, glandular stomach; Lane 6, intestine; Lane 7, colon; Lane 8, kidney; Lane 9, spleen; Lane 10, liver; Lane I I, testis; Lane 12, ovary; and Lane 13, uterus. (C) Lane I, S4 (glandular stomach adenocarcinoma); Lanes 2 and 3, m73 1 and m673 (choriocarcinoma); Lane 4, RRCC-I (renal cell carcinoma); Lane 5, normal esophageal tissue; Lane 6, esophageal squamous papilloma; Lane 7, esophageal squamous carcinoma; total RNAs prepared from Wistar fetal kidney (C. Lane 8), S-week-old Wistar adult kidney (C, Lane 9). and kidney carcinoma RRCC-I (C, Lane IO) were transferred onto the same filter to compare rSSRP1 mRNA levels in rat kidney directly. The blots were hybridized to the 32P-labeled rSSRPl cDNA probe. Hybridization withb-actin cDNA was used as the control. As a size marker, a 0.24-9.5 kb ladder (BRL) was coelectrophoresed.
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Fig. 3. Localization day 14 fetal Wistar digoxigenin-labeled
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of rSSRP1 mRNA in rat fetal kidney, adult kidney and renal cell carcinoma by in situ hybridization. Frozen sections of rat kidney (A), renal cell carcinoma (B) and 5-week-old adult kidney (C) were hybridized with the rSSRP1 antisense RNA probe, and day 14 fetal Wistar rat kidney with the sense probe (D). Original magnification xS0.
fetal kidney, adult kidney and renal cell carcinoma are shown in Fig. 3. Using the digoxigenin-labeled antisense RNA probe, rSSRP1 expression was demonstrated in the fetal kidney including epithelial cells of the comma-shaped body and s-shaped body (Fig. 3A), and in the neoplastic cells of renal cell carcinoma (Fig. 33). Expression of rSSRP1 was not detectable in any cells of 5-week-old adult kidney (Fig.
3C). No positive signals were observed In the sections hybridized with the digoxigenin-labeled sense rSSRP1 probe (Fig. 3D). 4. Discussion The SSRP family, a group of high mobility group (HMG) box-containing proteins, is considered to be a
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new class of transcription regulators [ 16-211. Although the ultimate function of the SSRP family has not been clearly determined, their structurerecognition properties are consistent with the hypothesis that their HMG-box domains facilitate the recognition of bent or bendable DNA structural elements. Human SSRPl binds to the drug cisdiamminedichloro-platinum(I1) (cisplatin)-modified DNA [ 161. Cisplatin is widely used in cancer chemotherapy and is especially effective in the treatment of testicular and ovarian cancer [22]. It forms covalent adducts with DNA that alter its structure and block replication and transcription [23]. Recently, several models of the role of HMG domain proteins in mediating the toxicity of cisplatin have been proposed [20,24,25]. The Drosophila homologue of human SSRPl, dSSRP1 [19], and yeast SSRP, Ixr 1 [20], also bind to cisplatin-modified DNA. Another member of the SSRP family, mouse protein T160, has been shown to bind to signal sequences for V-(D)-J recombination [ 181, and the homologous rat protein (CIIDBP-r) [17] was isolated by screening a chondrosarcoma cDNA expression library with a sequence located in the first intron enhancer of the collagen II gene, which has been shown to bind chondrocyte nuclear factors
PII. Up to now, the SSRP family genes have been investigated from the standpoint of their capacity to recognize a particular DNA structural alteration. The RNA expression of SSRP family genes in rat and mouse tissues was unclear. Human SSRPl is strongly expressed in adult baboon brain and spleen, and weakly in heart, ileum, jejunum, kidney, liver and muscle. It is also expressed in several bladder and testicular carcinoma cell lines [ 161. There is no more information available on its expression in fetal and tumor tissues. Northern blot analysis of a variety of rat organs, including fetal, adult and tumor tissues, in our experiment revealed particular expression of rSSRP1 in rat fetal kidney and Fe-NTA-induced kidney carcinoma, but no expression in adult kidney. This result was further demonstrated by in situ hybridization. rSSRP1 was also detected in some fetal and adult tissues and a few tumor tissues, but no specific expression was found in any other organs examined. Although the location of rSSRP1 protein should be further clarified with antibody, the specific
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pattern of rSSRP1 expression in the developing kidney and Fe-NTA-induced kidney carcinoma suggests that it must play a significant role in both morphogenesis and tumorigenesis. The mechanistic basis of this observation remains to be characterized. The molecular changes in DNA induced by FeNTA have been widely investigated both in vitro and in vivo [6-lo], but the chemical nature of those products has not been sufficiently characterized. Recent studies indicate that some of the changes are DNA intrastrand cross-links [ 10,261. It is interesting that the major cisplatin-DNA adducts recognized by SSRPl are 1,2-intrastrand d(GpG) and d(ApG) crosslinks [27], although we were unable to demonstrate the presence of intrastrand cross-linking in kidney DNA from Fe-NTA-treated rats and determine whether it has the same structure as the cisplatinmodified DNA. Comparison of the entire predicted amino acid sequences in human SSRPl, mouse T160 and the Drosophila homologue reveals 48% identity [19]. The broad evolutionary conservation among SSRP family members indicates that they perform a critical cell function. Furthermore, the pleiotropic expression pattern of SSRP1[16] and rSSRP1 seems to imply a more general function for these proteins. Evidence has accumulated that HMG domain proteins other than SSRP family members [ 16,19,20] also recognize cisplatin-modified DNA [25,28-311. HMG domain proteins appear to play important roles in DNA replication, transcription and repair [32]. Lack of a clearly defined consensus sequence among the HMG-box domains in these proteins [16] may indicate that such proteins have both non-specific and specific DNA-binding properties. Although we cannot exclude the possibility that expression of rSSRP1 reflects the active proliferative potential of both fetal and cancerous cells, the specific expression of rSSRP1 in rat kidney led us to speculate that the common DNA structural alterations may occur in both Fe-NTA-induced renal cell carcinoma and fetal kidney during kidney development. In view of the DNA-binding activity of the SSRP family, we assume that whatever the alterated structure of the DNA, it should contain DNA-binding components for SSRP. Our findings should add useful information on the DNA alterations in Fe-NTA-induced renal cell carcinoma.
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Acknowledgements We are very grateful to Dr. Makoto Miyamoto, Department of Pathology, Osaka University Medical School, Japan, for supplying the rat tumor cell lines. This work was supported in part by the Ministry of Education, and the Ministry of Health and Welfare of Japan. References [I]
Mottola, H.A. (1974) Nitrilotriacetic acids as chelating agent: applications, toxicology, and bioenvironmental impact. Toxicol. Environ. Chem. Rev., 2.99-161. [2] Anderson, R.L., Bishop, W.E. and Campbell, R.L. (1986) A review of the environmental and mammalian toxicology of nitrilotriacetic acid. Crit. Rev. Toxicol., 15, t-102. [3] Goyer, R.H., Falk, H.L., Hogan, M., Feldman, D.D. and Richter, W. (1981) Renal tumors in rats given t&odium nitrilotriacetic acid in drinking water for 2 years. J. Natl. Cancer Inst., 66, 869-880. [4] Okada, S. and Midorikawa, 0. (1982) Induction of rat renal adenocarcinoma by Fe-nitrilotriacetate (Fe-NTA). Jpn. Arch. Intern. Med., 29, 485-491. [S] Li, J.L., Okada, S., Hamazaki, S., Ebina, Y. and Midorikawa, 0. (1987) Subacute nephrotoxicity and induction of renal cell carcinoma in mice treated with ferric nitrilotriacetate. Cancer Res., 47, 1867-1869. (61 Toyokuni, S. and Sagripanti, J.L. (1993) DNA single- and double-strand breaks produced by ferric nitrilotriacitate in relation to renal tubular carcinogenesis. Carcinogenesis, 14, 223-227. [7] Kawabata, T., Awai, M. and Kohna, M. (1986) Generation of active oxygen species by iron nitrilotriacetate (Fe-NTA). Acta Med. Okayama, 40, 163-173. [8] Toyokuni, S., Mori, T. and Dizaroglu, M. (1994) DNA base modifications in renal chromatin of Wistar rats treated with a renal carcinogen, ferric nitrilotriacetate. Int. J. Cancer, 57, 123--l 28. [9] Umemura, T., Sai, K., Takagi, A., Hasegawa, R. and Kurokawa. Y. (1990) Formation of 8-hydroxydeoxyguanosine (8. OH-dG) in rat kidney DNA after intraperitoneal administration of ferric nitrilo-triacetate (Fe-NTA). Carcinogenesis, II, 345.-347. [IO] Randerath. E., Watson, W.P., Zhou, G.D., Chang, J. and Randerath, K. (1995) Intensification and depletion of sptcific bulky renal DNA adducts (l-compounds) following exposure of male F344 rats to the renal carcinogen ferric nitrilotriacetate (Fe-NTA). Mutation Res., 341, 265-279. [I I] Xiang. Y.-Y., Tanaka, M., Suzuki, M., Igarashi, H., Kiyokawa, E., Naito, Y., Ohtawara, Y., Shen, Q., Sugimura, H. and Kino, I. (1994) Isolation of complementary DNA encoding K-cadherin, a novel rat cadherin preferentially expressed in fetal kidney and kidney carcinoma. Cancer Res., 54, 3034-304 I. [12] Okada, S.. Hamazaki, S., Ebina, Y., Fujioka, M. and Mi-
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dorikawa, 0. (1983) Nephrotoxicity and induction of the renal adenocarcinomaby ferric-nitrilotieeerare (Fe-NTA) in rats. In: Struclure and Function of Iron Stonrge and Transport Proteins, pp. 473-478. Editor: 1. Urushizaki. Elsevier, Amsterdam. [I31 Xiang. Y.-Y. Wang, D.-Y., Tanaka, M., Igarashi, H., Kamo, T., Shen, Q.. Sugimura, H. and Kino, I. (19951 Efficient and specific induction of esophageal tumms in rats by precursors of iV-nitrososarcosine ethyl ester. Pathot. Int.. 4.5,43 5-42 1. [I41 Sambrook, J.. Fritsch, E.F. and Mania&, T. (1989) Molecular Cloning: a Laboratory Manual (2nd edn.). Cold Spring Harbor Laboratory Press, New York. [I51 Tanaka. M., Sasaki, H.. Kino, I., Sugimura, T. and Tenda, M. (1992) Genes preferentially expressed in embryo stomach m predominantly expressed in gastric tanL%r. Cancer Res., 52, 3372.-3377. [I61 Bruhn, S.L., Pit, P.M., Essigmann, J.M., Housman, DE and Lippard, S.J. (E 992) Isolation and characterization of human cDNA clones encoding a high mobility group box protein that recognizes structural distortions of DNA caused by binding of the anticancer agent cisplatin. Proc Natl. Acad. Sci. USA, 89, :!307-23 11. [I71 Wang, L., Precht. P., Balakir, R. and Horton. Jr.. W.E. (1993) Rat and chick cDNA clones encoding HMG-like proteins. Nucleic Acid. Res., 21, 1493. [IS] Shirakata, M., Huppi, K., Usuda, S., Qkazaki, K., Yoshida, K. and Sakano, H. (1991) HMGI-related DNA-binding protein isolated with V-(D)-J recombination signal probes Mol. Cell. Biol., II, 4528-4536. [19] Bruhn, S.L., Housman, D.E. and Lippard, S..l, (1993) lsolation and characterization of cDNA clones encoding the 5r0sc$zilu homolog of the HMG-box SSRP family that recognizes specific DNA structures. Nucleic Acids Res.. 21 1643-1646. (201 Brown, S.J., Kellett, P.J. and Lippard, S.J. (1993) lxr 1, a yeast protein that binds to platinated DNA and confers sensitivity to cisplatin. Science, 261, 603-605. [21] Wang, L., Balakir, R. and Horton, W.E. (1991) ldentificstion of a &-acting sequence in the collagen II enhancer rcquired for chondrocyte expression and the hinding of a chondrocyte nuclear factor. J. Biol. Chem.. 366, 1987% 19881. [22] Loehrer. P.J. and Einhorn, L.H. (1984) Drugs five years later: cisplatin [Review]. Ann. Intern. Med.. 100, 704-7 13. [23] Bruhn, S.L., Toney. J.H. and Lippard, S.J. (1990) Biological processing of DNA modified by platinum compounds. Prog. Inorg. Chem.. 38, 477--5 16. 1241 Huang, J.C., Zamble, D.B., Reardon, J.T., Lippard, S.J. and Sancar, A. (1994) HMG-domain proteins specifically inhibit the repair of the major DNA adduct of the anticancer drug cisplatin by human excision nuclease. Proc Natl. Acad. Sci. USA, 91. 10394-10398. [25] Treiber, D.K., Shai, X., Jantzen, H.M. and Esslgmann, J.M. ( 1994) Cisplatin-DNA adducts are molecular decoys for the ribosomal RNA transcription factor hUBF (human npslream binding factor). Proc. Natl. Acad. Sci. USA. 91. 5672-5676. [26] Lesko, S.A., Drocourt, J.-L. and Yang, S.-U. (19821 Deoxyribonucleic acid-protein and deoxyrihonucl& acid inter-
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Expression of structure-specific recognition protein mRNA in fetal kidney and Fe-nitrilotriacetate-induced renal carcinoma in the rat Yun-Yan Xianga, Dong-Yu Wanga, Masamitsu Tanakaa, Hisaki Igarashia, Yasuhisa Naitoa. Yoshihisa Ohtawarab, Qiong Shenc, Haruhiko Sugimuraa,* “First Department of Pathology, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 43 l-31, Japan bDepartment of Urology, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-31, Japan ‘Departmeni qf Precancerous Studies, Henan Medical Universiry Zhengzhou, Henan 4.50052, People $ Republic, of China
Received22April 1996;revision received24 May 1996;accepted24 May 1996
Abstract
Specificexpressionof the structure-specificrecognitionprotein(SSRP)genewasinvestigatedin rat fetal, adult, and tumor tissuesusinga 2.0-kb partial sequence of rat SSRPcDNA isolatedfrom a cDNA library of rat renalcell carcinoma.The resultsrevealedthat it was rather specifically expressedin rat fetal kidney and renal cell carcinomainduced by Fenitrilotriacetate,but not in adult kidney,whenvariousorgansweretestedby Northernblot analysis.In situ hybridizationfurther demonstrated that it waslocatedin the neoplasticcellsof renalcell carcinomaand in the epithelialcellsof fetal kidney but undetectablein any cellsof normaladultkidney. Theseobservations seemto imply the involvementof SSRPgene,which is believedto recognizestructuralalterationsof DNA, in kidney developmentand carcinogenesis of certaintypes of kidney cancer. Keywords:
Rat kidney; Fetaldevelopment;Renalcell neoplasm;Structure-specificrecognitionprotein
1. Introdection Nitrilotriacetate (NTA) is an effective metalchelating agent used as a substitute for polyphosphates in laundry detergents [ 1,2]. Because chronic exposure to high levels of NTA producestoxicity and neopiasmsin the urinary tract [3], an experimental model of renal adenocarcinoma was established by repeated intraperitoneal injections of ferric nitrilotriacetate (Fe-NTA), an iron chelate of NTA that in* Corresponding author.Tel: +81 53 4352220;fax: +81 53 4352225.
0304-3835/96/$12.00 PI1
SO304-3835(96)04332-7
duces renal proximal tubular necrosis which in turn leads to a high incidence (@I-92%) of renal adenocarcinoma in male rats and mice [4,5]. The molecular changes in DNA induced by FeNTA have been investigated, and Fe-NTA has been found to induce DNA single- and double-strand breaks in the presence of reductant in vitro [6]. In recent years, the renal toxicity of Fe-NTA has come to be attributed to mediation by reactive oxygen species (ROS) [7-101 such as hydroxyl ra.dicals (OH) and hydrogen peroxide (H202). However, the only information available on altered gene expression in rat renal adenocarcinoma induced by Fe-NTA has
0 1996 Elsevier Science Ireland Ltd. All rights reserved
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been the overexpression of K-cadherin reported by us previously [l 11. In the present study, we obtained a cDNA clone that encodes the rat structure-specific recognition protein (SSRP) by screening a cDNA library of Fe-NTA-induced rat renal cell carcinoma. The expressions of rat SSRP in various rat organs were investigated by Northern blot analysis and in situ hybridization to see whether they are involved in kidney development and carcinogenesis. 2. Materials
and methods
2. I. Cell lines and tissues The transplantable tumor cell line RRCC-1 was derived from an AC1 rat renal cell carcinoma induced by Fe-NTA according to the method of Okada et al. [ 121. Other transplantable tumor cell lines, S4, derived from spontaneously occurring gastric adenocarcinoma of Wistar-Furth rats, and m731 and m673 derived from choriocarcinoma of Wistar-Furth rats were obtained from Dr. M. Miyamoto, Department of Pathology, Osaka University Medical School, Japan. Cells were maintained as described [ 111. Tissue from esophageal tumors induced by precursors of Nnitrososarcosine ethyl ester was obtained from Wistar rats [ 131. Tissue from various organs was obtained from day 20 fetal Wistar rats and 5-week-old Wistar rats. All of the samples were immediately frozen in liquid nitrogen and stored at -80°C until use.
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(T.Tn) obtained from the Japanese Cancer Research Resources Bank (JCRB). The hD-8 clone, cloned at the EcoRI site of the plasmid pBluescript SK(+) (Stratagene, LA Jolla, CA), is about 1.3 kb in size and extends from bp 725 to 2063 of the published human SSRPl nucleotide sequence [ 161 (Fig. 1). Northern blot hybridization analysis showed high level expression of hD-8 in the rat renal cell carcinoma RRCC-1. In the present study, therefore, the hD-8 cDNA clone was used as a probe for screening the RRCC-1 cDNA library to isolate rat cDNA for SSRP. To make the probe, plasmid DNA was digested with EcoRI and purified on a 1% agarose gel. 2.4. cDNA library construction
and screening
The rat RRCC-1 cDNA library was synthesized from 5 ,ug of cell line RRCC- 1 poly(A)+ RNA by the cDNA synthesis system plus an oligodeoxythymidylate primer (Amersham, Buckinghamshire, UK). The size-fractionated cDNA was ligated to ;Igt 10 arms and then packaged into phage particles with Gigapack II Gold Packaging Extract (Stratagene, LA Jolla, CA). The filters, prepared from the RRCC-I cDNA library, were screened with [a-32P]dCTPlabeled hD-8 cDNA probe. After hybridization, the filters were washed under a high stringent condition [ 141 and autoradiographed for 3 days. The cDNA clones isolated from the library were subcloned into the pGEM-T plasmid vector (Promega, Madison, WI).
2.2. RNA preparation 2.5. DNA sequencing Total RNAs of the cell lines, tumor tissues and various rat organs were extracted by guanidiniuml cesium chloride ultracentrifugation. The subsequent poly(A)+ RNA was then selected from total RNA by oligodeoxythymidylate cellulose chromatography [141. 2.3. Probe for screening In the previous study, we isolated a human D-8 cDNA clone (hD-8) from a human cDNA library by differential screening according to the method described previously [15]. The human cDNA library was established from a squamous cell carcinoma cell line (T.T) and its transplanted tumor in a nude mouse
Nucleotide sequences of the cDNA clones obtained from the RRCC-1 library were determined by the double-stranded dideoxy-chain termination method using T7 DNA polymerase (Sequenase; Version 2.0, United States Biochemical Corp., Cleveland, OH). A computer-assisted homology search was carried out by the FASTA program in the GenBank and EMBL databases using an on-line system to DDBJ (1111 Yata, Mishima, Japan). 2.6. Northern blot analysis Rat total RNAs (1Opg each) from various cell lines and tissues were separated in denaturing aga-
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rose gels and transferred to nitrocellulose membranes. Hybridization was carried out, followed by high stringent washing and autoradiography. Hybridization and washing conditions were as described by Sambrook et al. [ 141. 2.7. In situ hybridization In situ hybridization on cryostat sections was conducted as described previously [ll]. The rSSRP1 sense and antisense RNA probes were produced by in vitro transcription from linearized pGEM-T plasmids containing the entire rSSRP1 cDNA in both orientations in the presence of digoxigenin-labeled UTP (Boehringer Mannheim, Mannheim, Germany) using T3 or I7 RNA polymerase, according to the manufacturer’s instructions. Final washing conditions and detection of the hybridization signal were as described in [ 111. 3. Results 3.1. Isolation of a cDNA clone for SSRP A total of 3.5 X IO5 recombinant phages were screened with the initial human D-8 clone as a probe to isolate rat cDNA for SSRP. Three positive clones, which were demonstrated to be identical to each other by restriction enzyme mapping, were obtained after three rounds of purification. Computer-assisted comparison of the DNA sequences revealed one of them to be 100% homologous to the published SSRP cDNA sequences of rat CIIDBP-r [ 171 (Fig. 1, rCIhSSRP1
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Fig. 1. Diagrammatic nucleotide sequence comparison of rSSRP1 and hD-8 with known rat CIIDBP-r [17] and human SSRPl [16], respectively, and corresponding nucleotide positions. ICIIDBP, rat CIIDBP-r; hSSRP1, human SSRPl. The open boxes represent open reading fragments and the hatched box refers to the position of the HMG box in these nucleotide sequences.
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IDBP), which extends from 1 to 1353 bp of the whole CIIDBP-r sequence and contains 645 bp of additional 5’ sequencethat had 70% homology with the human SSRPl [16] and 94% homology with murine T160 [18]. Becauseit is isolated by using the partial sequenceof human SSRPl as a probe, we refer to the clone in this study as rat SSRPl (rSSRP1) (Fig. 1). 3.2. Expression of rSSRP1 mRNA in various rat tissues The mRNA Eevel of rSSRP1 was determined in various organs of fetal and adult rats using the entire length of rSSRP1 cDNA as a probe (Fig. 2). Total RNAs were isolated from the brain, heart, lung, stomach, intestine, colon, kidney, spleen, liver, skin, testis, ovary and uterus of 5-week-old Wistar rats and/or day 20 rat embryos. In the day 20 embryos, rSSRP1 was found to be strongly expressed in the brain, either heart or lung, stomach, spleen and kidney with a major transcript size of 2.9 kb (Fig. 2A, Lanes l-3, 6, 7). A low level of expression was also present in the liver, skin and placenta (Fig. 2A, Lanes4, 5, 8, 9). In adults, rSSRP1 was strongly expressedin the testis (Fig. 2B, Lane 11) and weakly expressed in the brain, heart, lung, forestomach, glandular stomach, colon, spleen, ovary and uterus (Fig. 2B, Lanes l-5, 7, 9, 12, 13)” but there was no expression in the intestine, kidney or liver {Fig. 2B, Lanes 6, 8, 10). Among the various organs investigated, tissuespecificity was only found in the kidney. rSSRP1 was strongly expressed in fetal kidney, but not expressed in adult kidney. Levels of rSSRP1 mRNA in neoplasmswere investigated by using kidney, esophagus,stomach and uterine tumor tissues. rSSRP1 was expressed in rat kidney transplantable tumor line RRCC-1 at the sametranscript size as in fetal kidney but not in adult kidney (Fig. 2C, Lanes 4, 8-10) whereasrSSRP1 expression was observed in both the cancer and normal tissuesof other organs including the esophagus(Fig. 2C. Lanes 5-7) stomach (Fig. 2B, Lanes4, 5, and C, Lane l), and uterus (Fig. 2B, Lane 13, and C, Lanes2, 3) 3.3.
In
situ hybridization of rSSRP1 in rat kidney
The results of in situ hybridization in rat day 17
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Fig. 2. Northern blot analysis of rSSRP1 in rat tissues. Total RNA (1Opg of each) was extracted from various organs of day 20 fetal Wistar rats (A), 5-week-old Wistar rats (B), and rat cancer tissues and cell lines (C). (A) Lane I, brain; Lane 2, heart and lung; Lane 3, stomach; Lane 4, intestine; Lane 5, liver; Lane 6, spleen; Lane 7, kidney; Lane 8, skin; and Lane 9, placenta. (B) Lane I, brain; Lane 2, heart; Lane 3, lung; Lane 4, forestomach; Lane S, glandular stomach; Lane 6, intestine; Lane 7, colon; Lane 8, kidney; Lane 9, spleen; Lane 10, liver; Lane I I, testis; Lane 12, ovary; and Lane 13, uterus. (C) Lane I, S4 (glandular stomach adenocarcinoma); Lanes 2 and 3, m73 1 and m673 (choriocarcinoma); Lane 4, RRCC-I (renal cell carcinoma); Lane 5, normal esophageal tissue; Lane 6, esophageal squamous papilloma; Lane 7, esophageal squamous carcinoma; total RNAs prepared from Wistar fetal kidney (C. Lane 8), S-week-old Wistar adult kidney (C, Lane 9). and kidney carcinoma RRCC-I (C, Lane IO) were transferred onto the same filter to compare rSSRP1 mRNA levels in rat kidney directly. The blots were hybridized to the 32P-labeled rSSRPl cDNA probe. Hybridization withb-actin cDNA was used as the control. As a size marker, a 0.24-9.5 kb ladder (BRL) was coelectrophoresed.
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Fig. 3. Localization day 14 fetal Wistar digoxigenin-labeled
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of rSSRP1 mRNA in rat fetal kidney, adult kidney and renal cell carcinoma by in situ hybridization. Frozen sections of rat kidney (A), renal cell carcinoma (B) and 5-week-old adult kidney (C) were hybridized with the rSSRP1 antisense RNA probe, and day 14 fetal Wistar rat kidney with the sense probe (D). Original magnification xS0.
fetal kidney, adult kidney and renal cell carcinoma are shown in Fig. 3. Using the digoxigenin-labeled antisense RNA probe, rSSRP1 expression was demonstrated in the fetal kidney including epithelial cells of the comma-shaped body and s-shaped body (Fig. 3A), and in the neoplastic cells of renal cell carcinoma (Fig. 33). Expression of rSSRP1 was not detectable in any cells of 5-week-old adult kidney (Fig.
3C). No positive signals were observed In the sections hybridized with the digoxigenin-labeled sense rSSRP1 probe (Fig. 3D). 4. Discussion The SSRP family, a group of high mobility group (HMG) box-containing proteins, is considered to be a
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new class of transcription regulators [ 16-211. Although the ultimate function of the SSRP family has not been clearly determined, their structurerecognition properties are consistent with the hypothesis that their HMG-box domains facilitate the recognition of bent or bendable DNA structural elements. Human SSRPl binds to the drug cisdiamminedichloro-platinum(I1) (cisplatin)-modified DNA [ 161. Cisplatin is widely used in cancer chemotherapy and is especially effective in the treatment of testicular and ovarian cancer [22]. It forms covalent adducts with DNA that alter its structure and block replication and transcription [23]. Recently, several models of the role of HMG domain proteins in mediating the toxicity of cisplatin have been proposed [20,24,25]. The Drosophila homologue of human SSRPl, dSSRP1 [19], and yeast SSRP, Ixr 1 [20], also bind to cisplatin-modified DNA. Another member of the SSRP family, mouse protein T160, has been shown to bind to signal sequences for V-(D)-J recombination [ 181, and the homologous rat protein (CIIDBP-r) [17] was isolated by screening a chondrosarcoma cDNA expression library with a sequence located in the first intron enhancer of the collagen II gene, which has been shown to bind chondrocyte nuclear factors
PII. Up to now, the SSRP family genes have been investigated from the standpoint of their capacity to recognize a particular DNA structural alteration. The RNA expression of SSRP family genes in rat and mouse tissues was unclear. Human SSRPl is strongly expressed in adult baboon brain and spleen, and weakly in heart, ileum, jejunum, kidney, liver and muscle. It is also expressed in several bladder and testicular carcinoma cell lines [ 161. There is no more information available on its expression in fetal and tumor tissues. Northern blot analysis of a variety of rat organs, including fetal, adult and tumor tissues, in our experiment revealed particular expression of rSSRP1 in rat fetal kidney and Fe-NTA-induced kidney carcinoma, but no expression in adult kidney. This result was further demonstrated by in situ hybridization. rSSRP1 was also detected in some fetal and adult tissues and a few tumor tissues, but no specific expression was found in any other organs examined. Although the location of rSSRP1 protein should be further clarified with antibody, the specific
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pattern of rSSRP1 expression in the developing kidney and Fe-NTA-induced kidney carcinoma suggests that it must play a significant role in both morphogenesis and tumorigenesis. The mechanistic basis of this observation remains to be characterized. The molecular changes in DNA induced by FeNTA have been widely investigated both in vitro and in vivo [6-lo], but the chemical nature of those products has not been sufficiently characterized. Recent studies indicate that some of the changes are DNA intrastrand cross-links [ 10,261. It is interesting that the major cisplatin-DNA adducts recognized by SSRPl are 1,2-intrastrand d(GpG) and d(ApG) crosslinks [27], although we were unable to demonstrate the presence of intrastrand cross-linking in kidney DNA from Fe-NTA-treated rats and determine whether it has the same structure as the cisplatinmodified DNA. Comparison of the entire predicted amino acid sequences in human SSRPl, mouse T160 and the Drosophila homologue reveals 48% identity [19]. The broad evolutionary conservation among SSRP family members indicates that they perform a critical cell function. Furthermore, the pleiotropic expression pattern of SSRP1[16] and rSSRP1 seems to imply a more general function for these proteins. Evidence has accumulated that HMG domain proteins other than SSRP family members [ 16,19,20] also recognize cisplatin-modified DNA [25,28-311. HMG domain proteins appear to play important roles in DNA replication, transcription and repair [32]. Lack of a clearly defined consensus sequence among the HMG-box domains in these proteins [16] may indicate that such proteins have both non-specific and specific DNA-binding properties. Although we cannot exclude the possibility that expression of rSSRP1 reflects the active proliferative potential of both fetal and cancerous cells, the specific expression of rSSRP1 in rat kidney led us to speculate that the common DNA structural alterations may occur in both Fe-NTA-induced renal cell carcinoma and fetal kidney during kidney development. In view of the DNA-binding activity of the SSRP family, we assume that whatever the alterated structure of the DNA, it should contain DNA-binding components for SSRP. Our findings should add useful information on the DNA alterations in Fe-NTA-induced renal cell carcinoma.
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Acknowledgements We are very grateful to Dr. Makoto Miyamoto, Department of Pathology, Osaka University Medical School, Japan, for supplying the rat tumor cell lines. This work was supported in part by the Ministry of Education, and the Ministry of Health and Welfare of Japan. References [I]
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